The aim of this proposal is to study the regulatory mechanisms of liver cell proliferation using primary cultures of adult rat liver parenchymal cells. Our previous studies defined hormonal conditions for the stimulation of DNA replication and cell division in cells from partially hepatectomized livers. Combinations of insulin, epidermal growth factor (EGF) and glucagon, at physiological concentrations, acted synergistically to stimulate incorporation of labeled thymidine into morphologically distinct hepatocyte nuclei. We have now extended these studies to cells from intact rat livers. We have optimized conditions for culturing these cells and find similar hormonal requirements as with the cells from partially hepatectomized livers. In the presence of the 3 hormones the cultured cells enter DNA synthesis on the third day and peak on the fourth day. We will now begin studies into the early events in the hormonal stimulation of cell growth. Insulin, EGF and glucagon will be characterized with respect to their temporal requirements in the sequence of events leading to DNA synthesis. We will then determine the effects of hormone mixtures and of the individual hormones on cAMP and cGMP levels, and on their ability to affect nuclear protein phosphorylation.